The objective of this proposal is to produce homozygous cell lines from existing hemizygous transgenic goats and cows, for use in producing homozygous transgenic animals. GTC Biotherapeutics (formerly Genzyme Transgenics Corp) has developed a safe, abundant and cost effective production source for recombinant biopharmaceuticals by expressing recombinant proteins in the milk of transgenic dairy animals. Current transgenic methods produce first generation hemizygous animals that carry the transgene(s) at a single chromosomal integration site. Doubling the transgene copy number by producing homozygous animals would double protein productivity and simplify future breeding schemes. However, the only existing way to generate homozygotes is through two generations of progeny testing. We have devised a strategy for rapidly selecting homozygous primary cell lines that are derived from the original transgenic animals and that can be used as nuclear transfer donors to produce homozygous transgenic animals. We now propose to establish the feasibility of this strategy by: 1) optimizing methods for producing homozygous fibroblast cell lines; 2) characterizing the cell lines to confirm the presence of one transgene copy on each of two homologues; and 3) producing homozygous transgenic embryos by nuclear transfer. If this approach is successful, it will cost -effectively boost transgenic productivity.